Automated protein sequencing has become increasingly sensitive and compatible for analysis of blotted proteins. Vertical flow, blot cartridges in protein sequencers produce usable sequence information at the 10-20 pmole range which amounts to 0.5-1 .mu.g for a 50 kDa protein. Despite the increasing sensitivity of newer sequencing instruments, the isolation of unknown proteins often presents a technical challenge, particularly when only protein mass is known and specific antibodies or affinity ligands are unavailable.
The separation power of two dimensional polyacrylamide gel electrophoresis (2-D PAGE) has often been exploited as part of isolation schemes for determining the amino acid sequence of unknown proteins from complex protein mixtures. Proteins are usually elecrotransferred to inert membranes, detected with reversible stains which do not interfere with Edman degradative chemistries, excised and sequenced. Several purification procedures incorporate 2-D PAGE for protein isolation and sequencing. A common approach involves partial purification of the desired protein using any number of chromatographic methods, which commonly include molecular sieving, ion exchange and ligand- or immunoaffinity purification prior to 2-D PAGE as a final purification step. However, these methods involve extensive sample preparation and multi-step procedures which often rely upon following a known biological activity of the protein or adding trace amount of purified protein during the fractionation process.
Another strategy is to combine individual proteins from multiple, high-resolution 2-D PAGE isolation under analytical conditions (.ltoreq.100 .mu.g load). For example, Bauw et al (J. Electrophoresis, 1990 11, 528-536) pooled individual Coomassie Brilliant Blue (CBB)-stained proteins from 15-20 analytical 2-D PAGE gels into a single well for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). Each protein was electroeluted from multiple, 2-D PAGE gel punches into a single band during SDS-PAGE, electroblotted onto PVDF, stained and sequenced. However, the low sensitivity of reversible stains which do not interfere with sequence makes detection difficult for low abundance proteins. Such proteins in low concentrations are often only visible with sensitive metal stains or radiolabel. Also, protein loss during sample consolidation frequently limits the effectiveness of protein pooling for sequencing from multiple 2-D PAGE gels.
A third approach involves detection of critical protein changes by analytical 2-D PAGE and scale-up to preparative 2-D PAGE for isolation of sufficiently quantities of protein desired from sequencing. This approach has recently been used for sequencing two isoforms of phospholipase C-.alpha. induced by estrogen treatment or serum-induced proliferation (Mobbs et al., Science 1990, 247, 1477-1479; Merrice et al. Biochem. Arch. 1993, 9, 335-340). Another group has used preparative 2-D PAGE to sequence proteins from bacterial ribosomes and the postsynaptic density of bovine brain (Walsh et al, Biochemistry 1988, 27, 6867-6876; Walsh et al. J. Neurochem. 1992, 59, 667-678). These studies indicate the potential of preparative 2-D PAGE in identifying proteins perturbed by experimental treatment or those proteins which are selectively expressed in specific tissues.
At the present time, commercial devices for analytical 2-D PAGE are not readily adaptable for preparative sample loads from complex protein preparations in the milligram range. Although several preparative isoelectric focusing devices are available and have high sample capacity, they function as stand-alone units which are not immediately integrated into additional systems for final protein resolution and electrotransfer.
Strategies are needed for rapid protein isolation in order to identify disease-related proteins and facilitate the design of oligonucleotides for further molecular inquiry.